The incidence of serious fungal infections has increased considerably in recent decades. The "azoles" ketoconazole and itraconazole are important and widely-used oral antifungal agents, but the use of these drugs is limited by adverse side effects and serious drug-drug interactions. The antifungal activity of the azoles is based on the inhibition of a fungal cytochrome P450 (CYP) enzyme, and the problems of drug-drug interactions are the result of the inhibition of human CYP, mainly CYP3A4, by these azoles. Ketoconazole and itraconazole are chiral, and ketoconazole is used clinically as the racemic mixture of the two enantiomers, while itraconazole is a mixture of two racemates, i.e. four stereoisomers. The fundamental goal of this proposal is to define effective antifungal agents based on separating the stereoisomers of the two drugs and identifying the isomers with significantly reduced risks of drug interactions. Thus, one of the goals of the proposal is to elucidate the stereoselectivity of the in vitro inhibition of human CYP3A4 by the stereoisomers of ketoconazole and itraconazole. Microsomes from lymphoblasts expressing recombinant human CYP3A4 will be used to define the Ki and IC50 of the various stereoisomers using testosterone as substrate. Another goal is to determine the antifungal activity of the stereoisomers using standard microbroth dilution assays to establish minimum inhibitory concentrations (MIC's), and to evaluate stereoisomeric differences in the inhibition of the fungal CYP (CYP51). The stereoselectivity in the pharmacokinetics of ketoconazole and itraconazole in healthy subjects will also be evaluated after administration of the stereoisomeric-mixture-form in clinical use, both after initial drug administration and after reaching steady state. The stereoselective aspects of the metabolism of itraconazole to its active metabolite hydroxyitraconazole by microsomes from lymphoblast expressing recombinant human CYP3A4 will be examined. Results from the latter studies will provide important information on the processes that define the stereoselective pharmacokinetics of itraconazole. Finally, a series of analogs of ketoconazole will be synthesized for the evaluation of the importance of steric and electronic factors in stereoselectivity. Overall, the proposed studies will provide the foundation for the future administration of the individual stereoisomers of ketoconazole and itraconazole to patients with fungal infection. There is a need for new, safer azole antifungal agents, and the proposed studies will, by addressing the fundamental aspects of the stereoisomerism of the two drugs, define efficacious antifungal agents that are less prone to drug interactions based on differential inhibition of human and fungal CYPs.